1. Field of the Invention
The present invention relates to an analytical element for quantitative analysis of bilirubin, and more particularly to an analytical element employable for quantitative analysis of bilirubin based on a dry process operation with high accuracy by a simple and quick operation.
2. Description of Prior Arts
Bilirubin, a principal component of a bile pigment in a body fluid, is produced in serum by decomposition of heme originating from hemoglobin in red blood corpuscle. Bilirubin is then absorbed by a liver, in which bilirubin is converted to a glucuronic acid-conjugated product, etc. and excreted in bile. The content of bilirubin in blood increases in response to increase of decomposition of hemoglobin as well as decrease of the liver function. Accordingly, the quantitative analysis of bilirubin is considered to be an indispensible test item in the clinical test.
As the method for quantitative analysis of bilirubin in serum, there are known a quantitative analysis method comprising photometric measurement of the yellow color inherently attached to the bilirubin, and a colorimetric analysis of red azobilirubin produced by coupling reaction of bilirubin and diazotized sulfanilate (p-sulfobenzenediazonium salt, Ehrlich reagent) based on Ehrlich reaction discovered by Van den Bergh. The latter method is named a diazo method.
Details of methods for quantitative analysis of bilirubin in serum are described in "Comprehensive Text of Clinical Test Technology" edited by Ishii, Vol. 6, pp. 332-350 (Igaku Shoin, 1975).
Details of the diazo method are further described below.
Bilirubin produced in serum by the decomposition of heme is named free bilirubin. This bilirubin is as such hydrophobic, but is dissolved in serum in combination with serum albumin, being adsorbed by the serum alubmin. The free bilirubin introduced into liver is combined with glucuronic acid through covalent bond to become conjugated with glucuronic acid. Thus, a glucuronic acid-conjugated bilirubin which is enhanced in the water-solubilitiy by the aid of the hydrophilic group contained in the glucuronic acid is produced. Also known is a highly water-soluble bilirubin combined to serum albumin, but no production mechanism is known on this produce (J. J. Lauff, et al., Clinical Chemistry, 28(4), 629-637 (1982)).
Among these various bilirubins, the highly watersoluble conjugated bilirubin and the albumin-conjugate bilirubin both easily react with a diazonium salt, and are directly subjected to colorimetry. Accordingly, these bilirubins are named direct bilirubins.
The hydrophobic free bilirubin undergoes coupling reaction in the presence of a reaction accelerator such as caffeine, sodium benzoate, sodium acetate, dyphylline (C. A. Registory No. [479-18-5]), urea, a nonionic surfactant, gum arabic, an alcohol (e.g., methanol, ethanol), an acid amide, sulfoxide, etc., to produce axobilirubin. Therefore, the quantitative analysis of free bilirubin is generally performed indirectly by a stage of colorimetrically determining the total bilirubin content in a liquid sample in the presence of a reaction accelerator and a subsequent stage of subtracting the direct bilirubin content determined separately in the absence of a reaction accelerator from the total bilirubin content. For this reason, the free bilirubin is otherwise named an indirect bilirubin.
Details of the diazo method for quantitative analysis of bilirubin are described in the following publications: M. Michaelsson, Scand. J. Clin. Lab. Invest., 13 (Suppl.), 1-80 (1961); H. Malloy, J. Biol, Chem., 119, 481(1939); and Z. K. Shihabi, et al., American Journal of Medical Technology, 43(10), 1004-1007(1977).
As for the diazonium salt employed in the bilirubin analysis based on the diazo method, improvements have been recently made with respect to the detection sensitivity and stability of the produced azobilirubin. For instance, halobenzenediazonium salts such as 2,4-dichlorophenyldiazonium salt and 2-chloro-4-nitrophenyldiazonium, and stabilized diazonium salts (stabilized by the use of counter ions) developed by Kulhanek, Erthinghansen, et al. are generally utilized. The history of such development is understood, for instance, by referring to Japanese Patent Publication No. 54(1979)-12840, and Japanese Patent Provisional Publications Nos. 55(1980)-4492, 56(1981)-10255, 56(1981)-12555 and 57(1982)-103056.
As described above, a colorimetric analysis method comprising performing a color reaction in proportion to the content of an analyte (substance to be analyzed) and subsequetnly measuring the color formation to determine the content of the analyte is well known. This method is utilized not only in a wet analysis process but also in a dry analysis process.
The dry process (i.e., dry analysis process) is based on a colorimetric analysis utilizing a dry analytical element in the form similar to the pH test strip, which comprises a paper sheet or absorbent carrier impregnated with a reagent to produce a color in contact with an analyte.
As the dry analytical element, there is known a multilayer analytical element capable of giving highly precise analytical result. For instance, multilayer analytical element described in Japanese Patent Publication No. 53(1978)-21677 (corresponding to U.S. Pat. No. 3,992,158), and Japanese Patent Provisional Publications Nos. 50(1975)-137192 (U.S. Pat. No. 3,983,005), 51(1976)-40191 (U.S. Pat. No. 4,042,335), 52(1977)-3488 (U.S. Pat. No. Re. 30,267), 53(1978)-89796 (U.S. Pat. No. 4,069,017), 53(1978)-131089 (U.S. Pat. No. 4,144,306), etc. are in the form of a laminated structure comprising a support, one or more reagent layers on the support, and a porous, nonfibrous spreading layer on the reagent layer.
The above-mentioned multilayer analytical element is constructed in such a manner that a liquid sample applied (for instance, spotted) on the spreding layer permeates into the reagent layer, keeping a substantially constant amount per a unit area, and shows therein a color reaction. Accordingly, the content of the analyte in the liquid sample can be determined by measuring the color density after the lapse of a certain period of time.
A multilayer analytical element for quantitative analysis of bilirubin based on the dry process is already known. This element utilized a color reaction between bilirubin and diazotized sulfanilate (p-sulfobenzenediazonium, a bilirubin detection reagent) in the reagent layer thereof.
However, since the above-mentioned diazonium salt is highly hydrophilic and of high polarity, some problems are brought about into the analytical process employing the multilayer analytical element. For example, in the course of diffusion of the liquid sample into the reagent layer after spotting the liquid sample on the spreading layer of the analytical element, the diazonium salt is liable to be distributed ununiformly through the so-called chromatographic behavior to reduce the uniformal distribution of the azobilirubin showing color.
Moreover, the diazonium salt is liable to diffuse between the layers in the course of the preparation and storage of the multilayer analytical element, whereby the accuracy of the bilirubin analysis decreases as the time progresses. This means that the effectively employable period of the analytical element is shortened.
Into the multilayer analytical element, certain improvements have been introduced for enhancing the accuracy of the measurement. For instance, a light-blocking layer and an isotropically porous spreading layer are provided to the element. Otherwise, the diazonium salt is locally located in the analytical element. Even in thus improved multilayer analytical elements, the above-mentioned low molecular weight diazonium salt contained in the reagent layer is very liable to diffuse into the light-blocking layer and spreading layer, in the course of the preparation and storage thereof. The high diffusive prorperty of this diazonium salt is considered to arise from its low molecular weight. In the multilayer analytical element containing thus diffused diazonium salt, bilirubin contained in a liquid sample spotted thereon produced a not a small amount of azobilirubin even within the light-blocking layer and spreading layer. Accordingly, the total bilirubin produced by bilirubin and the diazonium salt cannot be quantitatively measured by a refelection optical measurement. In other words, the measured value shows negative error.